In many fields of life sciences research, including biological, biomedical, genetic, fermentation, aquaculture, agricultural, forensic and environmental research, there is a need to identify nucleic acids, qualitatively and quantitatively, in pure solutions and in biological samples. Such applications require a fast, sensitive, and selective methodology that can detect minute amounts of nucleic acids in a variety of media, whether or not the nucleic acid is contained in cells.
Although certain unsymmetrical cyanine dyes were first described before the genetic role of nucleic acids was established (Brooker, et al., J. AM. CHEM. SOC. 64, 199 (1942)), some unsymmetrical cyanine dyes are now known to be effective fluorescent stains of DNA and RNA. The cationic cyanine dye sold as Thiazole Orange has particular advantages in reticulocyte analysis (U.S. Pat. No. 4,883,867 to Lee, et al. (1989) and No. 4,957,870 to Lee et al. (1990)). Thiazole Orange readily stains many mammalian cells, yet does not effectively stain some eukaryotic cells.
Attachment of various cyclic structures to the pyridininm or quinolinium ring system of selected unsymmetrical cyanine dyes was found to make these nucleic acid stains highly permeant to gels and a wider variety of cell types, including both Gram-positive and Gram-negative bacteria, yeasts, and eukaryotic cells as well as prokaryotic cells, as described in international publication WO 94/24213 (Oct. 27, 1994) corresponding to international application PCT/US94/04127 (Apr. 1, 1993)
Attachment of a cationic side chain at the nitrogen of the pyridinium or quinolininm ring system of the unsymmetrical cyanine dyes, on the other hand, was shown to make the stains relatively impermeant to all cells, except cells, particularly mammalian cells, where cell membrane integrity was destroyed, as described in UNSYMMETRICAL CYANINE DYES WITH CATIONIC SIDE CHAINS (U.S. Pat. No. 5,321,130 to Yue et al. (1994)). A second type of dye, in which a dye monomer is attached at the nitrogen of the quinolinium or pyridininm ring system to form dimeric compounds as described in DIMERS OF UNSYMMETRICAL CYANINE DYES (PCT 92/07867) and DIMERS OF UNSYMMETRICAL CYANINE DYES CONTAINING PYRIDINIUM MOIETIES (U.S. Pat. No. 5,410,030 to Yue, et al., 1995) that is also relatively impermeant to all cells unless the cell membrane has been disrupted. The high sensitivity of nucleic acid detection made possible by these dyes, however, indicates that the affinity, and therefore sensitivity, of the stain corresponds to the increased cationic charge of these unsymmetrical cyanine dyes.
Unlike all of the cyanine dyes described above, the core structure of all of the dyes of the present invention is electrically neutral. Limited examples of these dyes have been previously described (Hamer, J. CHEM. SOC., 799 (1940); Clark, J. CHEM. SOC., 507 (1936)). Although these dyes are useful for the present invention, neither the complex of these dyes with nucleic acids, the use of these cyanine dyes to stain nucleic acids, nor the fluorescence properties of the dyes with or without nucleic acids has been described previously. In particular, the use of the dyes of the present invention to stain the nucleic acids of living cells has not previously been described. Surprisingly, the dyes of the current invention possess unexpected advantages as nucleic acid stains.
First, a formally neutral nucleic acid stain would not be expected to possess a useful affinity for nucleic acid polymers. As described above, the presence of additional cationic charges on already charged cyanine dyes has been shown to lead to higher affinity and sensitivity. Virtually all currently used nucleic acid stains possess at least one permanent positive charge. A formally neutral dye would not be expected to possess sufficient affinity to be a useful nucleic acid stain, because a significant part of the dye-nucleic acid binding energy is expected to be derived from the electrostatic attraction of the cationic dye to the negatively charged nucleic acid polymer. Nevertheless, the instant dyes, for which fluorescence depends on binding to nucleic acid polymers, are sometimes more fluorescent than Thiazole Orange.
In addition, the presence of a positive charge on nucleic acid stains currently used in the art enhances the ability of these dyes to enter cells. Nevertheless, the dyes of the present invention have exhibited utility as live cell stains, particularly when used to stain reticulocytes. As shown in Example 23, the dyes of the invention exhibit uptake rates in live cells comparable to, or faster than, that of Thiazole Orange. In addition, some dyes of the invention are useful in differentiating reticulocytes from other blood cells and blood components.
Further, the dyes of the present invention possess utility as electrophoresis gel stains and in detecting nucleic acids in solution. When viewing a DNA gel using illumination of 254 nm, the limit of detection using Thiazole Orange is 1-2 ng/band. In contrast Dye 510 of the present invention has a detection limit of 180 pg/band, and Dye 798 of the present invention has a detection limit of 350 pg/band (Example 22). These results indicate a utility for detection of nucleic acids that would not be expected in view of the performance of Thiazole Orange.